Because of its low toxicity and good therapeutical
performance, traditional Chinese medicine has attracted considerable attention
for many fields. Rhododendron dauricum L., referred to as ?Man-shan-hong?
in Chinese, belongs to the family of Ericaceae, the genera of Rhododendron L.
Rhododendron dauricum L. is distributed in the northern part of China, and
is a type of medicinal herb often used to cure chronic tracheitis. Chinese
Pharmacopoeia (1977 edition) has listed R. dauricum as an official drug
(1). Flavonoids are a kind of natural organic compounds with potential
physiological activities and effective components of Chinese Herbs, which have
attracted more and more research interest and exist in nature extensively.
Numerous flavonoids have been reported in the leaves and twigs of R. dauricum.
They are mainly composed of farrerol, flavonone, flavonol, flavone and flavonol
glucoside (1). Pathological experiments have showed that farrerol moves phlegm
and alleviates coughs, and farrerol has been synthesised chemically as an
antibechic (2, 3). (Besides moving the phlegm and alleviating cough, other
related investigations have shown that flavonoids could have a broad range of
physiological activities such as anti-inflammatory, anti-bacterial and
antioxidant activity for scavenging radicals).

The mechanisms of R. dauricum flavonoids (RF)
on respiratory system have been investigated, in which a decrease in [Ca2+]i
of bronchial smooth muscle seems to be involved. It is also well known that
intracellular calcium-induced calcium release (CICR) is involved not only in
bronchial and vascular smooth muscle, but also in myocardial cells, which could
induce the smooth muscle and myocardial cell constriction. It has also been
reported that [Ca2+]i overload causes cell damage, as a
result of cell undergoing hypoxic and ischemia. Should RF be capable of
inhibiting influx of Ca2+ and intracellular calcium release in order
to reduce [Ca2+]i, it would be able to avoid damage by
calcium overload.

Hence, the present study was designed to examine
the effects of on the vascular tone and explore its cardiovascular protective
effects, both in vivo and in vitro.

Experimental

Extraction of RF

Rhododendron dauricum (300 g) was extracted
twice at 80-100?C, using 3000 mL water for 2 h each time. The extracts were then
combined and filtered through filter paper. The filtrate was concentrated at
50-60?C by rotary evaporator to 1800 mL. This crude extract was then dissolved
in three volumes 95% ethanol, under constant stirring. The ethanol suspension
was laid overnight and then concentrated. Polyamide column chromatography
(volume of 400 mL, and a speed of 400 mL/h) was used to enrich the flavonoid
components. Non-flavonoid components were first eluted with 1200 mL distilled
water and then 1500 mL 95 % ethanol was used to elute the flavonoid components,
until reaching a negative reaction of hydrochloric acid-magnesium. The elution
speed was about 400 mL/h. The flavonoids suspension was concentrated and then
vacuum dried. The flavonoids powder was about 6 g and included hyperoside,
quercetin, azaleatin and isorhamnetin.

Male Wistar rats were purchased from Harbin Medical
University animal center. The rats (each weighing 250 - 300 g) were sacrificed
by decapitation. The thoracic aortas were rapidly and carefully dissected and
placed into ice-cold and oxygen-saturated K-H buffer solution (pH=7.4)
containing 118 mmol/l NaCl, 4.7 mmol/l KCl, 1.1 mmol/L MgSO4, 1.2
mmol/L KH2PO4, 1.5 mmol/L CaCl2, 25 mmol/L
NaHCO3, and 10 mmol/L glucose. The thoracic aortas were cleaned of
connective tissue, and cut into approximately 3-4 mm-wide strips. For
endothelium-intact strips, extreme care was taken to avoid endothelium injury.
For endothelium-denuded strips, the endothelium was removed by rubbing the
vessel interior with wet filter paper. Each vascular strip was mounted in a 5-mL
organ bath containing K-H buffer solution. One end of the aortic strip was
attached to a metal hook and the other end was connected to an isometric force
transducer (JH-2, metrical range: 0-10.0 g) in a bath containing K-H buffer
solution, maintained at 37?C and bubbled with 95% O2 and 5% CO2.
Rings were equilibrated for 45 min at 1.5 g resting tension, and then challenged
with PE (1.0?10-5 M) until a maximal contractile response was
obtained. The integrity of the endothelium was assessed in all preparations by
determining the ability of carbachol (1.0?10-5 M), to induce more
than 80% relaxation of rings. The endothelium was considered to be removed when
there was less than 10% relaxation response to carbachol. The isometric tension
was recorded with MedLab BL-420E+ recording system (Chengdu TME Technology Co.,
China).

Effects of RF on vascular tone

The experimental set up were consisted of 4 groups,
with each group including six test animal.

When the tension was at resting state or reached a
plateau induced by PE (1.0?10-5 M) or KCl (60 mM), RF (0.5, 1, 2, 3,
4 g/L) was cumulatively added into the organ bath at 4 min intervals. The rings
with intact or denuded endothelium were always tested in parallel.

Aortic rings were pretreated with RF (2 mg/mL) in
K-H buffer solution without Ca2+ 30 min before contraction with PE.
Then PE (1.0?10-5 M) was added into the organ bath at 4 min intervals
to examine the role of RF on PE- induced contraction.

Preparation of rat cardiomyocytes

Myocytes were isolated according to the method
established by Yang et al. (5). Briefly, each rat was killed, then the heart was
quickly removed and cannulated on a Langendorff apparatus and retrogradely
perfused through the aorta with the standard Tyrode?s solution (126 mM NaCl, 5.4
mN KCl, 10 mN HEPES 0.33 mN, NaH2PO4?2H2O 1.0
mN, MgCl2?6H2O, 1.8 mN CaCl2 and 10 mN glucose;
pH=7.4) for 5 min, and calcium-free Tyrode?s solution until it stopped beating.
The heart was then enzymatically digested with the calcium-free Tyrode?s
solution, containing collagenase type II and BSA. The ventricular tissue was
minced after being softened and placed in the KB medium (70 mM glutamic acid, 15
mN taurine, 30 mN KCl, 10mN KH2PO4, 10 mN HEPES, 5.4 mN
MgCl2?6H2O, 10 mN glucose and 0.5 mN EGTA; pH=7.4). Single
cells were obtained by gentle pipetting. It was stored at 4?C for 1?2 h, and
then gassed with 95% oxygen and 5% carbon dioxide and warmed to 37 ? 0.5?C. Only
rod-shaped myocytes with clear cross-striations were studied.

[Ca2+]i measurement

After the isolation of single ventricular myocytes,
they were adhered to the cover-slips of the chamber. Cells were then rinsed once
with the normal Tyrode?s solution and subsequently incubated with a working
solution containing Fluo-3/AM (20 mM) and Pluronic F-127 (0.03%) at 37?C for 45
min. After loading, the cells were washed once with the standard Tyrode?s
solution to remove the extracellular Fluo-3/AM. Fluorescent changes of the
Fluo-3/AM-loaded cells were detected by a laser scanning confocal microscope at
488 nm for excitation from an argon ion laser and 530 nm for emission and
inverted microscope with 20 ? objective. Drugs were added between scans 3 and 4,
and the images stored on disks. The fluorescent intensities, both before (F0)
and after (Fi) the drug administration, were recorded. The change in
[Ca2+] i was represented by the ratio of F i /F0.

Effect of RF on cardiomyocytes

The experiments were carried out on 3 groups, each
consisting of twenty test animal.

The preparation was pretreated with RF (1~2 mg/mL)
for 10 min. F i was then measured after adding 60mN KCl.

(ІІІ) Effect of verapamil on [Ca2+]i
elevation induced by KCl (60 mM)

The preparation was pretreated with calcium channel
inhibitor verapamil (10 μM) for 10 min. F i was then measured after
adding 60 mM KCl.

Effect of RF on hypoxia mice

Fourty male mice (average weight of 20?2 g) were
divided into 4 groups: 180 mg/kg RFH group, 90 mg/kg RFL group, control group
(normal saline) and 10 mg/kg BN52021 group. Each group received intraperitoneal
injection with isoprenaline (0.05 mg/10 g) 15 min after injecting drugs through
vena caudalis and then each animal moved into a wide-mouthed bottle (180 mL)
containing 10 g nitrica calx to examine the survival time, through the
observation of asphyxia.

Rat model of myocardial infarction

Male Wistar rats weighing 230?250 g were randomly
divided into five groups: control, ischemia (MI), ischemia-low RF (RFL, 7.5
mg/kg), ischemia-high RF (RFH, 30 mg/kg) and BN52021 (10 mg/kg), respectively.
The rats were heparinized (300 U) and anesthetized with IP sodium pentobarbital
(40 mg/kg), and were then ventilated using a small animal ventilator at a
frequency of 70/min and a tidal volume of 3 mL. The chest was surgically opened
and the body temperature was maintained at 37?C by placing the animal on a
heating pad. The standard limb lead ECG, together with the arterial pressure,
was continuously recorded on a recorder (Nihon Kohden RM 6200; Tokyo). A left
thoracotomy was performed via the fourth rib intercostal space and a segment of
saline-soaked 5-0 suture was looped around the left anterior descending (LAD)
coronary artery, near its origin from the left coronary artery and then the
chest was closed quickly.

Measurements of infarction size

The heart was removed from the animal after 60 min
of infarction, and then the ventricular tissue was dissected and kept at −4?C
overnight. The frozen ventricles were sliced into 2 mm thick sections, and then
incubated in 1% triphenyltetrazolium chloride at 37?C in 0.2 M Tris buffer
(pH=7.4) for 30 min. While normal myocardium was stained brick red, the
infarcted areas remained unstained. The size of the infarcted area was estimated
by its volume and weight, as a percentage of the left ventricle (6).

Statistical analysis

Data were presented as mean ? SD. Statistical
comparison was performed by the Student?s t - test and with an analysis of
variance (ANOVA), with a value of p < 0.05 considered as significant.

Results

Effects of RF on isolated aortic rings induced by
PE and KCl

RF relaxed PE (10-5 M)-precontracted
aortic rings in a dose-dependent manner (Figure 1). Compared with endothelium -
denuded rings, the maximal relaxant effect of RF on rings with endothelium was
increased by about 20 ? 1.8% and RF also relaxed KCl (60 mM)-precontracted
aortic rings in a dose-dependent manner. Compared with endothelium-denuded
rings, the maximal relaxant effect of RF on rings with endothelium was increased
by about 22 ? 1.9% (Figure 1).

The relaxant effect of RF on rings with endothelium
was obviously attenuated after pretreated with L-NAME (10-5 M) for 30
min and the maximal relaxant effect was degraded by about 22 ? 1.6%, close to
the endothelium-denuded rings (Figure 2).

The relaxant effect of RF on rings without
endothelium was obviously attenuated after pretreated with voltage dependent K+
channels (KV) inhibitor 4-AP and calcium activated K+ channels (KCa)
inhibitor TEA for 30 min, and the maximal relaxant effect was degraded by about
42 ? 1.5% and 21 ? 2.1% , respectively, compared with the control group. The ATP
sensitive K+ channels (KATP) inhibitor glybenclamide (Gli) and the
inward rectifier K+ channels inhibitor BaCl2 did not play a role on
the relaxant effect of RF, compared with the control group (Figure 3).

Effect of RF pretreatment in Krebs solution
without Ca2+ on Ca2+-induced contraction in isolated
aortic rings

The Ca2+- induced contraction and PE
induced contraction were obviously attenuated after pretreated with RF (2 mg/mL)
for 30 min in Krebs solution without Ca2+ containing 10-4 mol EGTA,
compared with the control group (Figure 4).

Effect of RF on cardiomyocytes [Ca2+]i

Prior to application of KCl, cardiac myocytes were
incubated with either 10 μM verapamil or different doses of RF. Both verapamil
and RF inhibited the increase in [Ca2+]i induced by KCl. At the time
point of 50s, KCl (60 mM) evoked a F i /F0 increase of 2.94 ? 0.11 fold, [Ca2+]i
(Figure 5). In contrast, in the group where cardiac myocytes were pre-incubated
with RF, KCl had modest effects on the increase in [Ca2+]i. However,
the inhibitory effect of RF on the increase in [Ca2+]i induced by KCl
was in a dose-dependent manner. As shown in Figure 6, the value of F i /F0 in
the presence of 1 mg/mL RF + KCl was 1.37 ? 0.04. In contrast, the value of F
i
/F0 in the presence of 2 mg/mL RF plus KCl was 1.06 ? 0.09 ( p < 0.05, n = 20)
(Figure 5).

Effect of RF on survival time of hypoxia mice

Compared with the control group, the mice survival
time of 180mg/kg RFH group and the positive control group were prolonged by 4
min and 6 min (p < 0.05), respectively. This finding suggested that RF could
obviously prolong the survival time in hypoxia mice (Table 1).

Effect of RF on cardial infarction size during
ischemia

The results, as shown in Figure 6, indicated that
the administration of RFL had a modest effect on the size of infarcted area of
rat heart (reduced by 4.32 %, p > 0.05). However, the size of infarcted area was
reduced by 16.14% at a higher concentration of RF (30 mg/kg). As a positive
control, 10 mg/kg of BN52021 was applied, which reduced the size of infarcted
area of heart by 17.52% (Figure 6).

Discussion

Rhododendron dauricum is a type of medicinal
herb often used to cure chronic tracheitis. The related literatures have
demonstrated that the flavonoids present in the leaves of R. dauricum
(such as Farrerol) could, relieve coughs and move phlegm. These flavonoids are
thought to reduce the [Ca2+]i , which can induce
relaxation of tracheal smooth muscle and decrease gland excretion. There are
several novel fingings in this present study: (i) the RF exerted a vasorelaxant
effect on the phenylephrine and KCl contracted aortic ring with or without
intact endothelium. (ii) Several mechanisms were involved, such as inhibition of
influx of extracellular Ca2+ and release of Ca2+ from
sarcoplasmic reticulum, contributing to NO release from intact endothelium,
activation of voltage dependent K+ channels (KV) and calcium
dependent K+ channels (KCa). (iii) In the rat myocardial cells,
incubation with RF could dose-dependently decrease [Ca2+]i
elevation by KCl, as observed by laser scanning confocal microscopy. (iv) In the
animal experiments, RF could significantly prolong the hypoxia endurance time in
mice and protect cardiac myocytes in the coronary artery ligation experiment.
Based on these findings, we concluded that flavonoids are the active components
of Rhododendron dauricum which can exert vasodilatation and myocardial
preservation. The potentials of RF on cardiovascular disorder are closely
related to a decreased [Ca2+]i content in vascular smooth
and cardiac muscle cells.

First of all, NO is a potent vasodilator
synthesized in the endothelium by the enzyme NO synthase, and causes vascular
smooth muscle cell relaxation through the activation of soluble guanylate
cyclase (7). The present study demonstrated that RF, dose-dependently, inhibited
the contraction induced by PE and KCl in intact aorta isolated from rats. This
vasorelaxant action was partially inhibited by pretreatment with L-NAME.
Endothelium-dependent relaxation of RF seemed to be associated with NO
signaling, via guanylate cyclase activation, since L-NAME could attenuate this
response. The direct endothelium-dependent and independent vasorelaxation should
be taken into account, based on this study. Secondly, RF dose-dependently
relaxed endothelium-denuded rings contracted with PE and KCl. The cellular
mechanism of contraction involved in the response to KCl and PE is different.
KCl induces Ca2+ influx via voltage-dependent Ca2+
channels, which further activates Ca2+-induced Ca2+
release through ryanodine-receptor. PE increases intracellular Ca2+
concentration via two mechanisms: (i) it activates receptor-gated Ca2+
channels, and (ii) it mobilizes Ca2+ from intracellular stores via
the inositol-1, 4, 5- trisphosphate (IP3) receptor or induces secondly myosin
light chain phosphorylation via activating PKC (8, 9). This means that, the
vasorelaxant action of RF seems to occur in both the receptor-dependent and
voltage-dependent manners in thoracic aortas. This hypothesis was confirmed
through further experiments, in which RF inhibited the dose-independent
contraction by CaCl2 and dose-independently decreased PE-induced
vasocontraction in Ca2+-free Krebs solution. Thirdly, many types of K+
channels have been identified in vascular smooth muscle cells (VSMC) (10), and
moreover, activated K+ channels induces hyperpolarization in order to
reduce the [Ca2+]i content in smooth muscle cells, which
can exert vasodilatation. The TEA, a calcium-activated K+ channel
inhibitor, and 4-AP, a voltage-dependent K+ channel inhibitor,
weakened the vasodilatory effect of RF. But glibenclamide, an ATP-sensitive K+
channel inhibitor, and BaCl2, an inward rectifier of K+ channel
inhibitor, had no influence on the vasodilatory effect of RF. It is concluded
that RF could activate the voltage-dependent K+ channel and the
calcium-activated K+ channel and encourage the K+ efflux
to reduce the [Ca2+]i content in smooth muscle cells.

In this study, isolated vascular rings experiments
indicated that RF-induced vasodilative effects partially contributed to the
inhibition of [Ca2+]i, and these facts prompted us to
testify the RF influence on isolated myocytes. Through the use of laser scanning
confocal microscope, we observed that RF could dose-dependently inhibit the
increase in [Ca2+]i induced by KCl, and the inhibitory
effect did not change with 10 μM verapamil at the concentration of 2 mg/mL. This
result confirmed that RF functioned to inhibit the [Ca2+]i
on both vascular smooth muscle cells and myocytes, which supported the
additional study, proposing that RF could exert cardioprotector effects. It has
been proven that reducing the [Ca2+]i content protects
myocytes during damage by hypoxia and ischemia (11, 12). The results of his
study indicated that RF prolonged survival time of hypoxic mice pretreated with
isoprenaline and reduced the myocardial infarction size in rat coronary artery
ligation, demonstrating that RF has a potential as a cardioprotector.

In conclusion, it could be said that explored the
cadiovascular potency of RF in isolated myocytes, and vascular tissue in this
study. Based on these studies, it could be said that so far the pharmacological
activities of R. dauricum exceed that of the traditional pharmacodynamic
limitations. Hence, RF seem to have the potential for development into a potent
drug for use in the cardiovascular system.

Acknowledgements

This work was supported by grants from the National
Natural Science Foundation of China (no. 30572181), key project of the National
Natural Science Foundation of China (no. 30430780) and the National Grand
Fundamental Research Pre-973 Program of China (no. 2007CB516803).